1. Field of the Invention
This invention relates to novel fluorescent proteins, GFPs and BFPs.
2. Related Background Art
GFP (Green Fluorescent Protein), which was found in Aequorea victoria, is a relatively small protein having a molecular weight of 26,900 and comprising the overall 238 amino acid residues as shown below (SEQ No. 1 in the Sequence Listing).
Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1         5            10          15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu       20           25           30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys     35            40           45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe   50            55           60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln             70           75            80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg          85          90           95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val        100          105          110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile     115          120           125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn  130          135            140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145           150            155           160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val           165            170           175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro       180           185           190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser     195          200           205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val  210           215          230 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225           230            235      238
In the present specification, the term “GFP protein” refers to a protein that emits green fluorescence when excited by ultraviolet-blue light and that, then, does not require an energy source such as a special substrate or ATP. In other words, the chromophore formation reaction of GFP is autonomous, and the portion of serine-tyrosine-glycine at Nos. 65–67 from the amino terminus forms an imidazolidine ring oxidatively which serves as a chromophore. (Yuichiro Watanabe, Gendai Kagaku “Modern Chemistry” 12, 46–52 (1995); R. Heim et al. Proc. Natl. Acad. Sci. USA 91:12501–12504 (1994).) Because GFP possesses such a property, a DNA encoding this protein is linked to a suitable expression vector and is introduced into the desired cells to express GFP, which alone results in fluorescent images. Therefore, GFP is in use for the visual analysis of gene expression and localization of proteins in a variety of cells in their viable state. However, since such GFP was not luminous at 37° C., there was a problem that culturing must necessarily be done at 30° C. for the purpose of observation in mammalian cells or the like. In connection with this problem, it has been reported that the mutations of V163A and S175G enhance the thermal stability. (K. R. Siemering et al. Curr. Biol. 6,1653–1663 (1996).)
Recently, a mutant of GFP into which the mutations of Y66H and Y145F were introduced and which had different wavelength characteristics (it is also referred to as “Mutant,” and its amino acid sequence (SEQ ID No. 15) is described below [with the above-mentioned mutations shown as underlined]) was developed. This is referred to as “BFP (Blue Fluorescent Protein),” because it emits blue fluorescence by UV excitation. (R. Heim et al. Curr. Biol. 6: 178–182 (1996); R. Heim et al. Proc. Natl. Acad. Sci. USA 91: 12501–12504 (1994).) In the present specification, the term “BFP protein” refers to a protein that emits blue fluorescence when excited by ultraviolet-blue light and that, then, does not require an energy source such as a special substrate or ATP. However, such BFP had a problem that it experienced severe fading as compared to GFP and was difficult to be observed under a microscope or the like. As used herein to designate mutation, the position of the mutation is expressed by a specific amino acid number in the sequence of the above-mentioned wild type; the amino acid prior to its mutation is described preceding the number and the mutated amino acid is to be described following the number.
Further, amino acids are designated by the one- letter code or three-letter code as appropriate.
(SEQ ID NO: 15)Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1         5            10          15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu       20           25           30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys     35            40           45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe   50            55           60 Ser His Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln   66         70           75            80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg          85           90           95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val        100          105          110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile     115          120           125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn  130          135            140 Phe Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145          150            155           160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val           165           170           175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro       180           185           190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser     195          200           205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val  210          215           230 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys.225           230            235      238